PROJECT SUMMARY Overall vision of the research program. My research is focused on understanding the ?molecular language? of cell-to-cell communication. Cell surface receptors transmit signals in response to external cues, and receptor activity is regulated by extrinsic factors such as posttranslational modifications, mechanical force, and chemical environment. Using a multidisciplinary approach that leverages my training in structural biology, biophysics, and protein engineering, my group is attempting to solve difficult problems in the area of receptor biology. We hypothesize that the distinct structural and biophysical parameters governing receptor-ligand interactions control downstream signaling, and that ligands may be engineered to alter these parameters and reprogram signaling outputs. My long-term goal is to harness the insight obtained through our mechanistic studies to design proteins that can fine-tune receptor activity and function as powerful next-generation therapeutics. Overview of research in the laboratory. Notch signaling is initiated when a mechanosensory Notch receptor is ?pulled on? by a Delta-like (DLL) or Jagged (Jag) ligand expressed on an adjacent cell. In mammals, the various Notch receptor (Notch1-4) and ligand (DLL1, DLL4, Jag1, Jag2) subtypes are able to induce distinct or even opposing cellular responses. Ligand selectivity is further regulated by Notch receptor glycosylation, which inhibits the activity of Jag ligands yet potentiates the activity of DLL ligands. We currently have a poor understanding of the molecular basis for ligand discrimination in the Notch pathway, and structural studies of Notch receptor-ligand interactions have been difficult because their nearly undetectable binding affinity precludes reconstitution of stable complexes. We recently overcame this obstacle by using directed evolution to strengthen DLL4 and Jag1 binding to Notch1, which allowed us to capture their interacting domains for x-ray crystal structure determination. My lab is now building upon these transformative results to explore how the structural, biophysical, and mechanical properties of various ligand subtypes contribute to their unique functions. Goals for the next five years. Over the five-year funding period, the goals of this project are to: (i) systematically characterize the structure, binding kinetics, and signaling potency of Notch receptor-ligand complexes; (ii) use electron microscopy to visualize the ultrastructural architecture of full-length Notch1-DLL4 and Notch1-Jag1 complexes; (iii) determine the biophysical basis by which Notch glycosylation regulates DLL and Jag activity, and (iv) design tissue-specific Notch antagonists. The successful completion of this work will establish the molecular mechanisms of Notch receptor-ligand selectivity and will guide the engineering of novel modulators of Notch signaling with expanded capabilities in a variety of biological and therapeutic contexts.